Regardless of when the manifestation occurs, the presence of one disease prevents the successful treatment of the other (Hasin et al., 1996). Finally, a number of factors, such as animal age and weight or time of day, can affect immobility time in the FST (Petit-Demouliere et al., 2005).
The Glutamate Hypothesis of Depression
In the prefrontal cortex (PFC), ketamine administration has been shown to rapidly and transiently activate the mTOR signaling pathway via AMPAR-dependent increases in phosphorylated, activated forms of p70S6 kinase, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and mTOR (Li et al., 2010). Furthermore, activation of glutamate synapses in the BNST stimulates negative affect-related behavior (Kim, et al. 2013).
GluN2B Knockdown in the BNST
Furthermore, we were able to demonstrate that targeted knockdown of the GluN2B subunit - the target of Ro 25-6981 and putative target of ketamine - specifically within the BNST produced a similar decrease in latency in the NIH test (Figure 2.1) with no effect on anxiety-like behavior measured by the EZM (Figure 2.S1). Perhaps an obvious future experiment in these animals would be to demonstrate a shutdown of ketamine's ability to further reduce latency in the NIH test, as has been shown with GluN2B deletion specifically of principal cortical neurons (Miller et al., 2014). However, the demonstration of lower possible latency values in the NIH task (as in Figure 1.1) reduces concerns about a floor effect phenomenon.
As discussed in Chapter 1, GluN2B-containing NMDARs are required for LTP induction in the BNST, as neither mice with constitutive forebrain-wide knockout of GluN2B nor slices acutely treated with Ro 25-6981 BNST show LTP (Wills et al., 2012). Preliminary data showed that ketamine (3mg/kg; 30 minutes before slicing) completely blocked LTP induction in BNST (Figure 4.1), as we expected. Finally, as described in the next section, it will be interesting to determine the effect of this regional deletion of GluN2B-containing NMDARs in other tests, such as the two bottle choice EtOH drinking test described in Chapter 3.
Implications of Depression-like behavior induced by protracted forced abstinence from a two-bottle choice EtOH drinking paradigm abstinence from a two-bottle choice EtOH drinking paradigm
Human AUDs are characterized by high levels of alcohol use and are highly comorbid with affective disorders such as depression and anxiety (Grant et al., 2015). In fact, of all patients admitted to hospital at risk of developing complicated AWS, only about 20% will experience these severe symptoms (Maldonado et al., 2015). Specifically, DID induced depression-like behavior as soon as 24 h after EtOH withdrawal (Lee et al., 2015).
Conversely, EtOH consumption can be increased through inhibition of FAAH (Hansson et al., 2007) or MAG lipase (Gutierrez-Lopez et al., 2010). Similarly, postmortem studies show reduced CB1 and FAAH in alcohol-dependent humans (Vinod et al., 2010). However, it was ineffective both in reducing consumption in non-treatment-seeking heavy drinkers (George et al., 2010) and in preventing relapse in treatment-seeking individuals (Soyka et al., 2008).
Future Directions utilizing the two-bottle choice EtOH drinking paradigm Sucrose two-bottle choice Sucrose two-bottle choice
Although CB1 agonists have been shown to increase EtOH recovery in rodents ( Lopez-Moreno et al., 2004 ), no change in EtOH recovery was observed following FAAH inhibition using URB597 ( Cippitelli et al., 2008 ). Two weeks post-surgery, mice were transferred to the Vanderbilt Murine Neurobehavior core and underwent the two-bottle choice EtOH paradigm with sucrose fade, as described in Chapter 3. Unfortunately, after initiating these studies, a report was brought to our attention which showed that AAV9-induced inflammation and neuronal loss (Ciesielska et al., 2013).
Our laboratory has shown changes in intrinsic excitability and plasticity in the BNST during acute withdrawal (~4 h) after CIE ( Kash et al., 2009 ; Wills et al., 2012 ). CIE exposure also inhibited the ability of acute EtOH to reduce NMDAR EPSC amplitude ( Kash et al., 2009 ). CIE increases LTP in the BNST through increased GluN2B expression, as CIE fails to increase LTP in forebrain-wide GluN2B knockout mice ( Wills et al., 2012 ).
Final Remarks
Future work will be necessary to move preclinical studies, including the work described above, into clinical treatments for suffering individuals.
Glucocorticoid Receptor-Mediated Effects within the Extended Amygdala Stress has been associated with a number of adverse effects, including anxiety disorders
2-AG binds to the cannabinoid receptor (CB1), leading to a decrease in presynaptic neurotransmitter release (Kano et al., 2009). This leads to a disinhibition of these projections to the NAc, and increased DA release within the NAc (Niehaus et al., 2010). Electrical stimulation of the CeA results in an HPA response with increased serum CORT (Weidenfeld et al., 1997).
The CeA contains the densest GR expression within the amygdala ( Morimoto et al., 1996 ), potentially implicating GRs in the CeA-mediated fear response. Selective pharmacological activation of GRs within the CeA elevates GR expression levels, increases anxiety-like behavior in the elevated plus maze (EPM), and increases plasma CORT in response to the stress of exposure to the EPM (Weiser et al., 2010). Chronic treatment with CORT has long been known to upregulate GR function within the brain (Spencer et al., 1991), leading to negative feedback impairment of the HPA axis.
Region-delimited deletion of GRs within the BNST do not alter affective behavior in mice
Extensive further research into the effects of GRs in the region on plasticity and anxiety and addictive behavior will prove crucial for a full understanding of such maladaptive responses to stress. Double IHC labeling of GR (red) and NeuN (green) reveals abundant GR expression in the BNST of (A) GR-floxed control mice and (B) significant reduction of GR expression in GR-floxed mice with BNST-targeted stereotaxic injection of LV-Cre. Finally, because the BNST is involved in responses to chronic – but not acute – stress, we postulated that a chronic stress paradigm may be necessary for affective changes in BNSTGRKD mice.
We used a chronic restraint stress paradigm in which mice were restrained in a 50 ml conical tube with breathing holes for 1 hour per day for 10 days. Open arm time (D) measured in the EZM after acute forced swimming stress and (E) in the EPM after 10 days of swimming.
Assessment of Acute Cocaine-induced Anxiogenesis using EPM
5mg/kg; 10 minutes before cocaine) had an effect on the open arm time in the EPM. A five-day treatment protocol prior to behavioral experiments is standard for most studies conducted by the Winder lab. However, not all groups include manipulation in their paradigms, and often—even when manipulation was performed—it is not included in the methods section in many manuscripts.
I administered cocaine (20mg/kg) or saline daily for 10 days (as illustrated in the timeline in Figure B.2). 20–22 h before the test each day, mice received cocaine or saline with propranolol or saline pretreatment. Chronic cocaine treatment had no effect on anxiety-like behavior in the EPM or NIH tests.
Western Blot Analysis of GluN2B knockdown in BNST
Other Approaches to GluN2B Analysis in BNST
In situ hybridization of GluN2B in the BNST and D2R in the striatum. A) GluN2B antisense probe in the BNST failed to clearly label GluN2B positive neurons. AAV9 appeared to have strong GFP and tomato fluorescence, and because of this, we used this virus in the two-bottle choice experiment described in Chapter 4. These neurotoxic effects appear to be a hallmark of AAV9-GFP-injections. Cre in BNST, and therefore this virus cannot be used in future studies.
We have high expectations for future experiments using a GFP-Cre fusion protein using AAV2 or AAV5 serotypes, as these have not been shown to cause tissue damage. GFP and tomato red fluorescence in the BNST of Rosa26 mice following tomato. stereotaxic delivery of AAV1-eGFP, AAV1-eGFP-Cre, or AAV9-eGFP-Cre.
Assessment of the Impact of Pattern of Cocaine Dosing Schedule During Conditioning and Reconditioning on Magnitude of Cocaine CPP, Extinction, and
Finally, we examined the ability of reconditioning with saline or a descending dose of cocaine to extinguish cocaine CPP. Our results are consistent with a previous report finding that a descending dose of cocaine is as effective as saline in destroying preference for the cocaine-associated context. On the eighth day, approximately 72 h after the last conditioning, mice were tested for the expression of cocaine CPP (post-test 1).
Experiment 3: Reconditioning extinction training with a descending cocaine dose schedule attenuates stress-induced reinstatement of cocaine CPP. A SNK post hoc analysis reveals that reconditioning with a decreasing dose of cocaine (compared with saline) has a significant effect on cocaine prime-induced reinstatement (Figure D.5; P < 0.05). However, we found that even when the total cocaine dose in the FLD group (30 mg/kg total) was less than half of the total dosage in the FHD group (64 mg/kg total), the magnitude of cocaine-CPP ens.
Acute Ketamine Administration has Heterogeneous Effects in Unidentified BNST Neurons
When we looked at individual responses to ketamine in female cells (Figure E.1B), we saw a variety of response types, suggesting heterogeneous responses to ketamine administration. Given the diversity of responses observed after ketamine administration in unidentified BNST neurons, we felt that we needed to narrow our parameters within this region to identify the specific effects of ketamine. Implications of BNST CRF in depression-related behavior lead us to examine this population of cells in the region.
GluN2B is Expressed on the Majority of CRF Neurons
Unlike the hippocampus or prefrontal. cortex, which are relatively homogeneous in the cell types present, the BNST contains a variety of cellular profiles, differing in both protein expression and projection targets. Previous work attempting to visualize GluN2B in the BNST had been less than fruitful (see Appendix B), but a new fluorescent ISH technique with higher sensitivity and lower background, RNAscope, was recently described (Wang et al., 2012). However, the magnified nature of such an image provides insight into only a small portion of the BNST.
RNAscope visualization of GluN2B and CRF mRNA expression in the BNST (A) Representative 63X images after RNAscope for CRF and GluN2B in the BNST. Figures E.3A and E.3A' show the proportion of GluN2B and/or CRF expression in all cells delineated with DAPI under both magnifications. The presence or absence of CRF expression in GluN2B-positive cells is examined in Figures E.3B and E.3B'.
Ketamine Reduces CRF mRNA expression in the BNST
Restraint and stimulation of bed nucleus of the stria terminalis produce similar stress-like behavior. Chronic social stress in the visual cavernous system modulates stress-related gene expression in the bed nucleus of the stria terminalis. Stress-induced changes in anxiety-like behavior and adaptations in plasticity in the bed nucleus of the stria terminalis.
Acute reversible inactivation of the bed nucleus of stria terminalis induces antidepressant-like effect in the rat forced swim test. Role of noradrenergic projections to the bed nucleus of the stria terminalis in the regulation of the hypothalamic-pituitary-adrenal axis. A corticotropin-releasing factor pathway for ethanol regulation of the ventral tegmental area in the bed nucleus of the stria terminalis.
